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Tn5 transposase-mediated library preparation of single-cell cDNAs for RNA-seq

ABSTRACT: Deep sequencing of single cell-derived genomic DNA and/or cDNAs brings novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-Seq requires multiple steps, including shearing the target DNA/RNA and following sequential enzymatic reactions, which result in consequent sample loss and stochastic variation at each step. Such variation may significantly affect the output from sequencing. We have found that a new technique of library preparation using hyperactive Tn5 transposase for the next-generation sequencer of Illumina's platform provided high-quality libraries from 100ng of short-length (average 700~800 bp) single-cell level cDNA. This new method reduced the number of steps in the protocol, which resulted in improved reproducibility and reduced variation among the specimens.

ORGANISM(S): Mus musculus

PROVIDER: GSE38198 | GEO | 2012/05/25

SECONDARY ACCESSION(S): PRJNA167516

REPOSITORIES: GEO

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Tn5 transposase-mediated library preparation of single-cell cDNAs for RNA-seq

Project description:Deep sequencing of single cell-derived genomic DNA and/or cDNAs brings novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-Seq requires multiple steps, including shearing the target DNA/RNA and following sequential enzymatic reactions, which result in consequent sample loss and stochastic variation at each step. Such variation may significantly affect the output from sequencing. We have found that a new technique of library preparation using hyperactive Tn5 transposase for the next-generation sequencer of Illumina's platform provided high-quality libraries from 100ng of short-length (average 700~800 bp) single-cell level cDNA. This new method reduced the number of steps in the protocol, which resulted in improved reproducibility and reduced variation among the specimens. Two methods of library preparation (sonication, tagmentation with hyperactive Tn5 transposase) were compared in the case of RNA-Seq for single-cell level cDNA. Technical triplicates were used.

2012-05-25 | E-GEOD-38198 | biostudies-arrayexpress

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Project description:Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from sub-picogram amounts of cDNA. Comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, as naked Tn5 can be annealed to any oligonucleotide of choice, for example molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enables innovation in sequencing-based applications.

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Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol [polyA]

Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries form little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.

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Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol [i5i7]

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Project description:We performed a parallel analysis of commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se generates pronounced sequencing biases, and subsequent fragmentation and amplification steps lead to several-fold overrepresentation of these artefacts. Standard pre-bisulfite library preparation methods lead to a significantly biased genomic sequence representation and a marked overestimation of methylation levels. We have integrated a bias diagnostic tool in the Bismark package and propose that amplification-free and post-bisulfite procedures should become the gold standard for WGBS library preparation.

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Tn5 Transposase Applied in Genomics Research.

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